Characterization of Plasmodium Falciparum ATP-Dependent DNA

Limudomporn et al. Malar J (2016) 15:526 DOI 10.1186/s12936-016-1573-2 Malaria Journal RESEARCH Open Access Characterization of Plasmodium falciparum ATP‑dependent DNA helicase RuvB3 Paviga Limudomporn1, Saengduen Moonsom1, Ubolsree Leartsakulpanich2, Pattra Suntornthiticharoen3, Songsak Petmitr4, Michael Weinfeld5 and Porntip Chavalitshewinkoon‑Petmitr1* Abstract Background: Malaria is one of the most serious and widespread parasitic diseases affecting humans. Because of the spread of resistance in both parasites and the mosquito vectors to anti-malarial drugs and insecticides, controlling the spread of malaria is becoming difficult. Thus, identifying new drug targets is urgently needed. Helicases play key roles in a wide range of cellular activities involving DNA and RNA transactions, making them attractive anti-malarial drug targets. Methods: ATP-dependent DNA helicase gene (PfRuvB3) of Plasmodium falciparum strain K1, a chloroquine and pyrimethamine-resistant strain, was inserted into pQE-TriSystem His-Strep 2 vector, heterologously expressed and affinity purified. Identity of recombinant PfRuvB3 was confirmed by western blotting coupled with tandem mass spectrometry. Helicase and ATPase activities were characterized as well as co-factors required for optimal function. Results: Recombinant PfRuvB3 has molecular size of 59 kDa, showing both DNA helicase and ATPase activities. Its 2 2 2 2 helicase activity is dependent on divalent cations (Cu +, Mg +, Ni+ or Zn+ ) and ATP or dATP but is inhibited by high NaCl concentration (>100 mM). PfPuvB3 is unable to act on blunt-ended duplex DNA, but manifests ATPase activity in the presence of either single- or double-stranded DNA. PfRuvB3.is inhibited by doxorubicin, daunorubicin and netrop‑ sin, known DNA helicase inhibitors. Conclusions: Purified recombinant PfRuvB3 contains both DNA helicase and ATPase activities. Differences in proper‑ ties of RuvB between the malaria parasite obtained from the study and human host provide an avenue leading to the development of novel drugs targeting specifically the malaria form of RuvB family of DNA helicases. Keywords: Plasmodium falciparum, ATP-dependent DNA helicase, ATPase activity, Helicase activity, PfRuvB3 Background in a search for new drug targets with which to combat the Malaria is one of the most serious and widespread scourge of malaria. parasitic diseases of humans. Globally, in 2015 an esti- The availability of the complete genome sequence of mated 3.2 billion people were at risk of being infected Plasmodium falciparum, the causative agent of fatal with malaria parasites and contracting the disease, with malaria, has opened new avenues to identify genes approximately 214 million people becoming infected important for parasite survival. Many potential chemo- resulting in a mortality of 438,000 [1]. With the emer- therapeutic targets involved in various metabolic path- gence of drug-resistant parasites to all available anti- ways at different malaria parasite life stages have been malarials, control of malaria is becoming difficult [1, 2]. identified recently [3]. Among these, helicases constitute This has led to efforts in developing novel strategies and a highly conserved group of enzymes important in all aspects of nucleic acid metabolism, such as replication, recombination, repair, transcription and (RNA) stability *Correspondence: [email protected] [3–6]. 1 Department of Protozoology, Faculty of Tropical Medicine, Mahidol RuvB is an ATP-dependent DNA helicase with a University, 420/6 Rajvithi Road, Bangkok 10400, Thailand Full list of author information is available at the end of the article hexameric ring structure and its architecture has been © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Limudomporn et al. Malar J (2016) 15:526 Page 2 of 10 suggested to be related to those of members of ATPases (with a SmaI site (underlined) upstream of the start ′ associated with various cellular activities (AAA+) pro- codon) and 5 -CCGCTCGAGATTACTTGTACTTGA tein class [6]. In Escherichia coli, RuvAB helicase is an ATTTTCCG-3′ (with a XhoI site shown underlined) ATP-driven translocase that promotes branch migra- based on genome sequence of P. falciparum 3D7 (NCBI tion of Holliday junction and formation of heteroduplex database accession no. XM001350297.1). Thermocycling DNA [6], in addition to being essential for replication conditions were as follows: 95 °C for 5 min; followed by fork reversal at occurrences of DNA replication defects 35 cycles of 95 °C for 10 s, 61.5 °C for 30 s and 72 °C for [7]. Expression of E. coli ruvB genes is required for DNA 1 min; with a final step at 72 °C for 90 s. The PCR was car- repair and maintaining normal levels of cellular resist- ried out using Phusion® High-Fidelity DNA Polymerase ance to stress-induced mutagenesis, so their resulting (Thermo Scientific, MA, USA) and amplicon was puri- mutants are thereby defective in recombination [8, 9]. fied using Nucleospin® extract II kit (Macherey-Magel, In eukaryotes, RuvB proteins, such as yeast RvB1 and Düren, Germany), digested with SmaI and XhoI, and RvB2, are nuclear proteins indispensable for cell cycle then ligated to similarly digested pQE-TriSystem His- progression and RNA polymerase II-dependent tran- Strep 2 expression vector (Qiagen, Germany) using T4 scription [10]. In addition, RuvBs are involved directly DNA ligase. The presence of PfRuvB3 in the recombinant in the regulation of transcription of over 5% of yeast plasmid was further verified for its nucleotide sequence genes as essential components of a chromatin remodel- by BioDesign (Pathumthani, Thailand). ling complex determining genes regulated by the complex [11] and indirectly by recruiting the TATA-binding Heterologous expression of PfRuvB3 and purification protein (TBP) to the promoter and its impairment confer of recombinant protein growth defects [12], and they are essential for viability in Recombinant pQE-TriSystem His-Strep 2-PfRuvB3 plas- all model organisms including Saccharomyces cerevisiae mid was transfected into Mix & Go Competent Cells- [13], Drosophila melanogaster [14] and Caenorhabditis Strain JM109 (Zymo Research Corporation, CA, USA) elegans [15]. Mutations in the conserved ATP-binding and transformed cells were selected on LB agar contain- and hydrolysis motifs of RuvBs decrease viability of these ing 100 μg/ml of ampicillin at 37 °C overnight. Cultures organisms [11]. were inoculated into LB broth containing 100 μg/ml Given that RuvBs play essential roles in nearly all of ampicillin and grown at 37 °C until A600 nm reached aspects of nucleic acid metabolism, P. falciparum RuvBs 0.4-0.6, and then treated with 1 mM isopropyl β-d-1- should be no exception. Analysis of P. falciparum genome thiogalactopyranoside (IPTG) at 37 °C for 1 h with shak- database identifies at least three RuvB homologues, ing. Cells were harvested by centrifugation at 2000×g at namely, PfRuvB1, PfRuvB2 and PfRuvB3 [16]. PfRuvB3, 4 °C for 15 min, washed with buffer (50 mM NaH2PO4 located on chromosome 13, comprises of 1452 bp encod- pH 8.0, 300 mM NaCl and 10 mM imidazole) and re-sus- ing for a 483-amino acid protein [17]. Recombinant pended in buffer supplemented with protease inhibitor PfRuvB1 and PfRuvB2 containing both helicase and cocktail (complete™ ULTRA Tablets, Roche, Germany) ATPase activities, and have been heterologously pro- before being lysed by sonication. Following centrifu- duced in E. coli and their properties characterized [18, gation at 9500×g at 4 °C for 45 min, supernatant was 19]. However, recombinant PfRuvB3 shows only ATPase applied onto a Ni–NTA affinity column (Qiagen, Hilden, activity, unlike that of the purified parasite protein that Germany), which was washed with washing buffer contains both helicase and ATPase activities [19, 20]. It (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 20 mM imida- is possible that the reported conditions for cloning and zole and 1 mM PMSF) until no protein was detected in expression were not optimal for producing fully active washed fractions. Recombinant PfRuvB3 was eluted with enzyme. Here, conditions for generating PfRuvB3 dual elution buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl activity in E. coli were examined and the effects of a num- and 250 mM imidazole) and fractions collected were sub- ber of known helicase inhibitors on the recombinant jected to analysis SDS-PAGE. Fractions containing the enzyme were also evaluated. 6xHistidine-tag fusion protein were pooled and applied onto a 1 ml Strep-Tactin® Sepharose® column (Iba Life Methods Sciences, Germany), which was washed with 5 ml of Cloning of Plasmodium falciparum ATP‑dependent DNA buffer A (100 mM Tris–HCl pH 8.0, 150 mM NaCl and helicase gene (PfRuvB3) 1 mM EDTA) and recombinant PfRuvB3 was eluted with Full length PfRuvB3 from P. falciparum K1, a chlo- buffer A containing 2.5 mM desthiobiotin. The recombi- roquine and pyrimethamine-resistant strain from nant protein was concentrated using an Amicon® Ultra- Thailand [21], was PCR amplified using primers 15 filtration unit and stored in buffer B (50 mM NaH2PO4 5′-TCCCCCGGGGCATGAAGCTCGAAGAAG-3′ pH 8.0, 100 mM NaCl and 30% glycerol). Protein Limudomporn et al. Malar J (2016) 15:526 Page 3 of 10 concentration was measured using Bradford assay (Bio- or 4 (Table 1) to single-stranded circular M13mp18 (+) Rad, CA, USA) and protein purity was checked by DNA (New England Biolabs, MA, USA) in 20 mM Tris– SDS-PAGE. HCl pH 7.5 containing 10 mM MgCl2, 100 mM NaCl and 1 mM DTT, incubating at 95 °C for 5 min and then Western blot and tandem mass spectrometry analysis cooling down slowly to room temperature over a period Following SDS-PAGE protein was transferred onto nitro- of 1 h [22].

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